Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure

Abstract

1. Increases in intravascular pressure depolarize vascular smooth muscle cells. Based on the attenuating effects of Cl- channel antagonists, it has been suggested that swelling-activated Cl- channels may be integral to this response. Consequently, this study tested for the presence of a swelling-activated Cl- conductance in both intact rat cerebral arteries and isolated rat smooth muscle cells. 2. A 50 mosmol l-1 hyposmotic challenge (300 to 250 mosmol l-1) constricted rat cerebral arteries. This constriction contained all the salient features of a pressure-induced response including smooth muscle cell depolarization and a rise in intracellular Ca2+ that was blocked by voltage-operated Ca2+ channel antagonists. The hyposmotically induced depolarization was attenuated by DIDS (300 microM) and tamoxifen (1 microM), a response consistent with the presence of a swelling-activated Cl- conductance. 3. A swelling-activated current was identified in cerebral vascular smooth muscle cells. This current was sensitive to Cl- channel antagonists including DIDS (300 microM), tamoxifen (1 microM) and IAA-94 (100 microM). However, contrary to expectations, the reversal potential of this swelling-activated current shifted with the Na+ equilibrium potential and not the Cl- equilibrium potential, indicating that the swelling-activated current was carried by cations and not anions. The swelling-activated cation current was blocked by Gd3+, a cation channel antagonist. 4. Gd3+ also blocked both swelling- and pressure-induced depolarization of smooth muscle cells in intact cerebral arteries. 5. These findings suggest that swelling- and pressure-induced depolarization arise from the activation of a cation conductance. This current is inhibited by DIDS, tamoxifen, IAA-94 and gadolinium.

Figure 1. Responses of intact cerebral arteries to hyposmotic challenge

Representative diameter (A), V_m (B) and cytosolic Ca²⁺ (C) responses to a 50 mosmol l⁻¹ hyposmotic challenge (300 to 250 mosmol l⁻¹). Nisoldipine (1 μm), a voltage-operated Ca²⁺ channel antagonist, blocked the rise in cytosolic Ca²⁺. D, effects of Cl⁻ channel antagonists (DIDS, 300 μm; tamoxifen, 1 μm) on hyposmotically induced depolarization. Data are means +s.e.m.* Significant difference from 250 mosmol l⁻¹.

Figure 2. Hyposmotic challenge enhances an outwardly rectifying whole-cell current in isolated cerebrovascular smooth muscle cells

A, ramp pulses (−100 to +100 mV, 0.167 mV ms⁻¹, holding potential =−50 mV, conventional whole-cell) were applied before and during a 50 mosmol l⁻¹ hyposmotic challenge. B, the swelling-activated difference current obtained by subtraction of the currents shown in A. C, whole-cell currents (+100 mV) in isosmotic (300 mosmol l⁻¹) and hyposmotic (250 mosmol l⁻¹) media are maintained over time. Following a 6 min period in isosmotic medium, cells were either maintained in this bathing solution or exposed to a 50 mosmol l⁻¹ hyposmotic challenge. Data are means ±s.e.m.* Significant difference from the isosmotic current.

Figure 3. Chloride channel antagonists inhibit swelling-activated currents

Ramp pulses (see Fig. 2 legend) were applied before and during a hyposmotic challenge and in the presence of DIDS (300 μm; A), tamoxifen (1 μm; B), or IAA-94 (100 μm; C). D, peak outward (DIDS, n = 6; tamoxifen, n = 6; IAA-94, n = 7) current measured at +100 mV. Data are means ±s.e.m.* Significant difference from 300 mosmol l⁻¹; ** significant difference from 250 mosmol l⁻¹.

Figure 4. The V_rev of swelling-activated current shifts with E_Na but not E_Cl

Ramp pulses (see Fig. 2 legend) were applied before and during a hyposmotic challenge. E_Cl was shifted from +1 to +31 mV by lowering the Cl⁻ concentration from 113 to 33 mM in the hyposmotic bath. B, E_Na and E_Cl were shifted (E_Na from 0 to −42 mV; E_Cl from +1 to +40 mV) by lowering the NaCl concentration from 110 to 20 mM in the hyposmotic bath.

Figure 5. Basal current properties

A, the basal current V_rev shifts with changes in E_Na. E_Na was altered (from 0 to −33 mV) by lowering the Na⁺ concentration from 110 to 30 mM in the isosmotic bath (300 mosmol l⁻¹). B, hyper-osmotic (350 mosmol l⁻¹) bath solution reduces the basal current. Inset, summary data (n = 4) showing peak outward currents (+100 mV) in isosmotic (□) and hyper-osmotic (▪) solutions. * Significant difference from 300 mosmol l⁻¹. C, fractional block of the basal and swelling-activated current by DIDS (300 μm), IAA-94 (100 μm) and tamoxifen (1 μm). Fractional block was calculated by dividing the leak-subtracted current (+100 mV) in the presence of the channel blocker by that in the absence of the blocker. Leak was estimated by linearly extrapolating the residual current at −100 mV (in the presence of blocker) and over the voltage range. Basal and swelling-activated currents were monitored in isosmotic (300 mosmol l⁻¹) and hyposmotic (250 mosmol l⁻¹) baths, respectively (n = 4–7 per group). Data are means ±s.e.m.

Figure 6. Gd³⁺ inhibits swelling-activated responses

A, ramp pulses (see Fig. 2 legend) were applied before and after a hyposmotic challenge and in the presence of Gd³⁺ (30 μm). B, peak outward current at +100 mV (n = 7). C, effects of Gd³⁺ (30 μm) on hyposmotically induced depolarization (250 mosmol l⁻¹, n = 4) in intact cerebral arteries. Data are means ±s.e.m.* Significant difference from 300 mosmol l⁻¹; ** significant difference from 250 mosmol l⁻¹.

Figure 7. Gadolinium and tamoxifen block pressure-induced depolarization

Cerebellar arteries were pressurized to 15 or 60 mmHg and V_m responses to Gd³⁺ (30 μm; 15 mmHg, n = 3; 60 mmHg, n = 6) and tamoxifen (10 μm, n = 5) were examined. Data are means ±s.e.m.* Significant difference from 60 mmHg.